Inhibition Of Sodium Pump Research Articles

Endogenous sodium pump inhibitors in human urine further identification of inhibitors of Na-K-ATPase

We investigated the presence of endogenous Na-K-ATPase inhibitor(s), ie, ouabain-like factors (OLFs), in the urine of salt-loaded healthy subjects. For this purpose 24-h urine was collected on days 3, 4, and 5 of high sodium intake (> 30 g NaCl/day). The samples then were lyophilized. Redissolved urine concentrates were acidified (pH 3.5) and subjected to gelchromatography on a Sephadex G-25 column where the OLFs eluted in the post-salt fraction IV. When lyophilized fraction IV was rechromatographed on Sephadex G-10, OLFs with molecular mass (M(r) of approximately 400 eluted in a late fraction IV/8 separate from added ouabain, ouabagenin (or digoxin), which eluted shortly after void volume. With the subsequent reverse-phase HPLC of fraction IV/8 a polar OLF-1 eluted in fraction IV/8a after the void volume in the water phase and a more apolar OLF-2 eluted at 20% acetonitrile in fraction IV/8d. Only the more apolar OLF-2 cross-reacted with a digoxin antibody. By preparative thin-layer chromatography OLF-1 and OLF-2 were purified as single compounds with potent dose-dependent Na-K-ATPase inhibition and Ki-values approximating 1.5 x 10(-5) mol/L and 1.5 x 10(-4) mol/L, respectively. Mass-spectroscopy (MS) showed M(r) of 391 and 1H-NMR characterized the endogenous urinary apolar OLF-2 as a compound that is structurally totally unrelated to ouabain; infrared (IR) spectroscopy of OLF-1 and OLF-2 also revealed no similarity with ouabain.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of the Ca 2+ Pump by the Hypothalamic-Hypophysary Inhibitory Factor

We previously purified to homogeneity an endogenous sodium pump inhibitor from bovine hypothalamus and hypophysis that is different from digoxin or ouabain and studied the effects of this factor on the total Ca2+,Mg(2+)-ATPase activity of plasma membrane of synaptosomes. This factor inhibits the calcium pump and the total Mg(2+)-ATPase activity of these membranes with approximately the same K0.5 values of inhibition. The potency of this factor as an inhibitor depends on the membrane concentration in the assay medium. The inhibition of the magnesium-dependent ATPase activities of these membranes was of a noncompetitive type with respect to the substrate Mg(2+)-ATP and did not significantly shift the calcium dependence of the Ca2+,Mg(2+)-ATPase activity. We suggest that the calcium pump of the synaptosomal plasma membrane is inhibited by this factor through disruption of the lipid annulus; this inhibition could play a role in the control of calcium homeostasis by increasing the cytosolic free calcium concentration.

Digitalis-like factor and digoxin-like immunoreactive factor in diabetic women with preeclampsia, transient hypertension of pregnancy, and normotensive pregnancy

An endogenous sodium pump inhibitor, or digitalis-like factor (DLF), has been postulated to mediate essential hypertension. It may also play a role in preeclampsia. However, studies of this factor in hypertensive pregnancy have not provided consistent findings. Part of this may be due to the absence of subclassification of pregnant women with pregnancy-induced hypertension (PIH) when assessing these parameters. In this study we explored serum DLF and digoxin-like immunoreactive factor (DLIF) in insulin-dependent diabetic (IDDM) women with normotensive pregnancies or PIH, comparing them to each other and to nondiabetic pregnant women. Our results demonstrated that nondiabetic women with preeclampsia (PE, PIH with proteinuria) had significantly increased serum DLF and DLIF compared to normotensive pregnant women (NL BP). Women with transient hypertension of pregnancy (THP, PIH without proteinuria) had intermediate values (DLF. NL BP: 3.3 +/- 0.6, THP: 4.8 +/- 1.1, PE: 7.6 +/- 1.3% inhibition [Na,K]-ATPase, P < .05 ANOVA; DLIF. NL BP: 0.22 +/- 0.02, THP: 0.28 +/- 0.03, PE: 0.35 +/- 0.02 ng digoxin equivalents/mL, P < .05 ANOVA). Pregnant normotensive IDDM women had significantly higher serum DLF and DLIF activity than their nondiabetic counterparts (DLF. non-IDDM NL BP: 3.3 +/- 0.6 v IDDM NL BP: 8.8 +/- 1.2% inhibition [Na,K]-ATPase, P = .0008; DLIF. non-IDDM NL BP: 0.22 +/- 0.02 v IDDM NL BP: 0.31 +/- 0.02 ng digoxin equivalents/mL, P = .005).(ABSTRACT TRUNCATED AT 250 WORDS)

Sustained volume expansion and [Na,K]ATPase inhibition in chronic renal failure.

Hypotheses regarding the pathogenesis of volume-dependent hypertension have invoked an endogenous sodium pump inhibitor or digitalis-like factor (DLF) to link altered sodium homeostasis to the rise in blood pressure. Our goal was to develop a clinical protocol that achieved predictable, sustained volume expansion, with the premise that renal failure patients on peritoneal dialysis would increase intravascular volume, gain weight, and raise blood pressure (BP) in relation to measured increases in DLF. In a 5-day protocol, dialysis was kept constant but dietary NaCl and fluids were modified in 7 patients. DLF was measured as inhibition of [Na,K]ATPase. Likewise, the first 2 L of daily peritoneal dialysate (PD) was processed on HPLC and the eluate analyzed for DLF. The group achieved significant weight gain (WT) by day 3 (delta WT = 4.1 +/- 1.2 kg, P < .05). Likewise, mean arterial pressure (MAP) and plasma DLF activity increased significantly. All variables were highly correlated (DLF v WT: R = 0.88, P = .004; MAP v DLF: R = 0.82, P = .01; MAP v WT: R = 0.90, P = .003). Although a number of HPLC fractions contained agents that interacted with the assay, only one PD HPLC fraction (at 19.5 min) contained DLF activity that correlated with changes in MAP (R = 0.60, P = .002), and body weight (R = 0.67, P = .0003). We conclude that candidate DLF responds to sustained volume expansion and the relationship suggests that it could influence blood pressure. Moreover, the application of stringent criteria to the confusing array of factors in plasma that may affect assays for DLF appears to reduce the field dramatically, to a single candidate in this setting.

Interactions of anticholinesterases with achatina fulica acetylcholine responses and electrogenic sodium pump

The dose-dependent effects of the anticholinesterases, neostigmine and mycotoxin territrem-B, were determined on: (i) Cl −-responses of voltage clamped Achatina fulica neurons to microperfused acetylcholine; (ii) the 4 K +-induced outward currents evoked by an electrogenic sodium pump in the same neuron; and (iii) acetylcholinesterase activity of Achatina fulica ganglionic homogenates. Both compounds at low doses potentiated the peak acetylcholine responses. However, they had different effects at higher (> l μM) doses in that neostigmine now antagonized acetylcholine responses, while territrem-B still produced a maximal potentiation. At all doses neostigmine produced a dose-dependent inhibition of acetylcholinesterase activity. The cholinolytic effect of high doses of neostigmine was associated with the inhibition of 4K +-induced current in the same neuron, while territrem-B neither altered the K +-induced current nor antagonized acetylcholine responses. The cholinolytic effect of neostigmine was completely antagonized by the inhibition of electrogenic sodium pump by ouabain or by perfusion with K +-free solution. These results suggest that neostigmine at high concentrations inhibits the electrogenic sodium pump and that the cholinolytic effect of high doses of neostigmine is secondary to this action. Territrem-B, on the other hand, had no effect on the electrogenic sodium pump and had no effect on the neuronal membrane properties other than to inhibit acetylcholinesterase. Thus, territrem-B may be a useful tool for studying the interaction between acetylcholinesterase and acetylcholine receptors.

Plasma potassium may protect sodium pumps of toad hearts from an endogenous inhibitor.

Resibufogenin (3-hydroxy-14,15-epoxy-20,22-dienolide glycoside) is a potent sodium pump inhibitor present in toad toxin. It is present in the skin of the cane toad (Bufo marinus) at a concentration equivalent to ouabain of approximately 1 mM. Because toads, like other amphibians, have permeable skin, resibufogenin is also found in high concentrations in the blood. In the cane toad the blood concentration is estimated to be 1 microM (D. Lichtstein, S. Kachalsky, and J. Deutsch. Life Sci. 38: 1261-1270, 1986; D. Lichtstein, S. Samuelov, J. Deutsch, H. Xu, R. A. Lutz, S. S. Chernick and S. S. Chernick. Klin. Wochenschr. 65, Suppl. 8: 40-48, 1987), a concentration thousands of times that required to produce toxicity in humans (J. S. Flier, E. Matatos-Flier, J. A. Pallotta, and D. McIsaac. Nature Lond. 279: 341-343, 1979). In examining how the cane toad avoids inhibiting its own sodium pumps, work on the heart showed that 1) cane toads possess a similar number of cardiac sodium pumps as other vertebrates, and 2) normal plasma K+ levels completely prevent ouabain, and presumably resibufogenin, from binding to cardiac sodium pumps of the cane toad. Other species, i.e., rat (Rattus norvegicus) and salamander (Ambystoma mexicanum), did not show K+ protection of their cardiac ouabain binding sites up to normal plasma K+ levels. These species do not possess the high level of endogenous ouabain-like substance found in the toad. K+ demonstrated a capacity to protect the enzymatic activity of the toad heart sodium pumps from the inhibitory effects of ouabain.

Sensitive assay for sodium pump inhibition

Sensitive assay for sodium pump inhibition

Transmembrane ion movements elicited by sodium pump inhibition in Helix aspersa neurons.

1. Transmembrane ion movements upon sodium-pump inhibition were studied in identified neurons of the subesophageal ganglia of Helix aspersa. A two-microelectrode, voltage-clamp technique was used to measure transmembrane currents. Changes in intracellular Na+, K+, and Ca2+ concentrations were measured, in unclamped neurons, with Na(+)-sensitive microelectrodes, K(+)-sensitive microelectrodes, and with the fluorescent probe fura-2, respectively. 2. Inhibition of the sodium pump with ouabain (1 mM) elicited an increase in intracellular Na+ concentration, [Na+]i, at an initial rate of 0.42 +/- 0.05 mM/min (mean +/- SE; n = 27), and a membrane depolarization often followed by hyperpolarization. In cells clamped at -50 or -60 mV, ouabain produced an inward shift in membrane-holding current followed by an outward current usually having two components, transient and sustained, respectively. 3. Replacing external Na+ with either N-methyl-D-glucammonium or tetraethylammonium (TEA+) abolished both the ouabain-induced inward membrane current and the rise in [Na+]i, suggesting that Na+ was the charge carrier of the inward current. 4. Cd2+ (400 microM) reduced the rate of rise of the inward current by 60% and the estimated net Na+ flux by 47%. 5. The outward current was abolished by K(+)-channel blockers (10 mM TEA+ and 5 mM 4-aminopyridine or 10 nM apamin). Cd2+ (400 microM), a Ca(2+)-entry blocker, also abolished the outward current. 6. Inhibition of the sodium pump elicited a fall in [K+]i at an initial rate of 1.4 +/- 0.2 mM/min (n = 9 cells). 7. Upon inhibition of the sodium pump in neurons loaded with fura-2, [Ca2+]i increased from an estimated resting level of 147 +/- 37 nM to a maximum of 764 +/- 248 nM (n = 12 cells). 8. The rise in [Ca2+]i in the sustained presence of ouabain was transient, lasting 19.5 +/- 2.8 min, and could be prevented by removal of external Ca2+ before ouabain application or curtailed by removal of external Ca2+ during sustained ouabain exposure. The latter effect was not a consequence of exhaustion of caffeine-sensitive intracellular Ca2+ stores. 9. It is concluded that 1) the rise in [Ca2+]i upon Na(+)-pump inhibition requires the presence of external Ca2+, 2) the outward current observed upon pump inhibition is a Ca(2+)-activated K+ current flowing through apamin-sensitive channels, 3) the resting Na+ permeability involves a Cd(2+)-sensitive component, 4) a large fraction (approximately 30-60%) of the previously described ouabain-induced cell shrinkage may result from Ca(2+)-activated K+ efflux contributing to net solute and water loss.

Blunted natriuretic response to human urine extracts with Na +, K +-ATPase inhibiting activity in experimental cirrhosis

Human urine and plasma extracts contain a material that inhibits the enzyme Na+,K(+)-ATPase (the endogenous sodium pump) and produces natriuresis in the bioassay animal. This endogenous sodium pump inhibitor(s), also known as digitalis-like factor, is thought to be involved in sodium and extracellular fluid volume homeostasis. Increased urine and plasma sodium pump inhibiting activity have been reported in patients with cirrhosis and sodium retention. The aim of the study was to assess the renal response to i.v. administration (0.2 ml/min per kg bw for 10 min) of a human urine extract containing sodium pump inhibiting activity (28.5 nmol equivalent ouabain/ml) in eight conscious rats with cirrhosis and ascites and eight control rats. Baseline urinary excretion of Na+,K(+)-ATPase inhibiting activity was significantly higher in cirrhotic rats with ascites than in control rats (235 +/- 40 vs 91 +/- 16; p < 0.01). Human urine extract induced a significant (p < 0.05) increase in glomerular filtration rate in control (3.2 +/- 0.4 to 4.2 +/- 0.5 ml/min) and cirrhotic rats (3.0 +/- 0.3 to 4.0 +/- 0.5 ml/min). In control rats it also increased urinary sodium excretion (1.47 +/- 0.22 to 2.43 +/- 0.5 microEq/min, p < 0.01) and fractional sodium excretion (0.29 +/- 0.01 to 0.43 +/- 0.04%, p < 0.025). In contrast, in cirrhotic rats with ascites neither sodium excretion nor fractional sodium excretion was significantly affected. No changes were observed in plasma aldosterone and atrial natriuretic peptide concentrations in either group. These data suggest that in cirrhosis there is a renal resistance to the natriuretic effect of endogenous sodium pump inhibitor(s).

Ouabain releases striatal polyamines in vivo independently of N-methyl-D-aspartate receptor activation.

Intrastriatally infused ouabain (200 or 1,000 microM) markedly increased the extracellular levels of striatal spermidine and spermine in dialysis experiments in halothane-anesthetized rats. The effects of ouabain (1 mM) on spermidine release were rapid and unaffected by local infusion of the competitive N-methyl-D-aspartate (NMDA) antagonist 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP; 100 microM) or by systemically administered MK-801 (0.3 mg/kg i.p.), both of which treatments markedly inhibit the effects of intrastriatally administered NMDA. The peak effects of ouabain (1 mM) on spermine release were delayed with respect to those on spermidine release, or to the effects of NMDA, and were also insensitive to locally administered CPP (100 microM). However, systemically administered MK-801 (0.3 mg/kg i.p., 30 min before the striatal infusion of drugs), which totally inhibits the effects of NMDA, or CPP (10 mg/kg i.p.; 30 min before the striatal infusion of drugs) partially inhibited the effects of ouabain on spermine release, suggesting partial mediation of the delayed effects of ouabain on spermine release by indirect NMDA-receptor activation. Despite partial sensitivity of ouabain-induced spermine release to systemically administered NMDA antagonists, both spermidine and spermine can be released in vivo by sodium-pump inhibition, independently of NMDA-receptor activation.

Effect of endogenous digoxin-like factor and digoxin antibody on myocardial Na+, K+-pump activity and ventricular arrhythmias in acute myocardial ischaemia in rats

The aim was to study whether a circulating sodium pump inhibitor (endogenous digoxin-like factor) contributes to the genesis of early ventricular arrhythmias in acute myocardial ischaemia in rats. Effects of digoxin antibody (260 micrograms.kg-1) on the incidence of ventricular arrhythmias, plasma digoxin-like immunoreactivity (DELFIA immunoassay), Na+, K+, and Mg2+ ions, and activity of the ouabain sensitive Na+, K(+)-pump in different regions of myocardium have been studied in propranolol naive and propranolol pretreated rats exposed to acute coronary artery ligation. Adult male Wistar rats were divided into six experimental groups: (1) saline pretreated controls; (2) saline pretreated coronary artery ligated rats; (3) coronary artery ligated rats pretreated with 260 micrograms.kg-1 digoxin antibody; (4) propranolol pretreated controls; (5) propranolol pretreated rats with acute myocardial ischaemia; (6) rats with acute myocardial ischaemia pretreated with both propranolol and digoxin antibody. Acute myocardial ischaemia in saline pretreated rats was associated with a twofold increase of plasma digoxin-like immunoreactivity and ventricular arrhythmias, but did not lead to changes in myocardial Na+, K(+)-pump activity. Pretreatment of coronary artery ligated rats with digoxin antibody reduced the total duration of ventricular tachycardia and ventricular fibrillation during a 15 minute postligation period from 201 (SEM 34) to 46(18) seconds (p < 0.002) but did not alter activity of the myocardial Na+, K(+)-pump. In rats pretreated with propranolol, acute myocardial ischaemia was associated with a twofold inhibition of the Na+, K(+)-pump in left atrial and left ventricular myocardium, and with a 69% increase in plasma K+ concentration. Administration of digoxin antibody to propranolol pretreated coronary artery ligated rats in parallel with the antiarrhythmic effect prevented the increase in plasma K+ concentration and inhibition of Na+, K(+)-pump in the left atrial, but not the left ventricular myocardium. A circulating digoxin-like factor contributes to the pathogenesis of myocardial ischaemia induced ventricular arrhythmias. As propranolol pretreatment of coronary artery ligated rats inhibited the Na, K(+)-pump in myocardium, the inhibitory effect of endogenous digoxin-like factor on Na+, K(+)-ATPase was probably masked in propranolol naive animals by the stimulatory action of catecholamines on Na+, K(+)-ATPase described previously.

Regulation by membrane potential of phosphatidylinositol hydrolysis in pancreatic islets

In pancreatic islets stimulated with carbamylcholine (carbachol), hydrolysis of both phosphatidylinositol (PtdIns) and phosphatidylinositol bisphosphate (Ptd-InsP2) occurs and can be measured as the inositol monophosphates Ins(1)P1, or Ins(4)P1, respectively (Biden, T. J., Prugue, M. L., and Davison, A. G. M. (1992) Biochem. J. 285, 541-549). Our current aim was to establish whether these two events were independently regulated. Rats islets were labeled with either [3H]inositol or [3H]arachidonic acid for measurement of InsP1s by high performance liquid chromatography or diacylglycerol by TLC, respectively. The rise in Ins(1)P1 due to carbachol (1 min) was inhibited by 50% by concomitantly raising extracellular KCl ([K+]e) from 6 to 30 mM, thereby depolarizing the islets. Similar results, obtained in the absence of extracellular Ca2+, exclude the involvement of voltage-gated Ca2+ channels. Conversely, hyperpolarization, by lowering [K+]e to 3 mM, increased by 30% the rise in Ins(1)P1. In fact, over the [K+]e range of 3 to 48 mM, stimulated Ins(1)P1 accumulation was directly proportional to the calculated membrane potential. In contrast, raising [K+]e from 6 to 48 mM exerted no significant effect on carbachol-stimulated Ins(4)P1 levels, and both Ins(1)P1 and Ins(4)P1 were unaffected in the absence of carbachol. The rises in Ins(1)P1 (but not Ins(4)P1) were also inhibited by depolarization with the sodium pump inhibitor, ouabain, or the K+ channel blocker, tolbutamide. Stimulated diacylglycerol accumulation and insulin secretion (20 min) showed a biphasic dependency on [K+]e, being less pronounced at 6 mM than at either 3 or 30 mM KCl. This reflects a selective potentiation of PtdIns and PtdInsP2 hydrolysis, due, respectively, to hyperpolarization and the gating of voltage-dependent Ca2+ channels. The differential regulation of these two hydrolytic events is probably important for independent control of the activation of protein kinase C and Ca2+ mobilization and might play a role in modulating the secretory response in vivo.

In Vivo Accumulation of Sodium Pump Inhibitor by Normal and Uremic Erythrocytes

A uremic toxin fraction (fraction 2-3-1) in the middle molecular mass range (300-2000 Da) containing a sodium potassium inhibitor pump was studied. As fraction 2-3-1 was known to be present in minute quantities in urine and plasma, erythrocyte lysates were used as an alternative source. Results show that fraction 2-3-1 is more abundant in erythrocytes than in normal urine. In addition, the fraction is present in a greater quantity in uremic erythrocytes than in normal blood cells. It is concluded that fraction 2-3-1 is concentrated at the erythrocyte level in uremic patients.

Endogenous natriuretic factors 1: Sodium pump inhibition does not correlate with natriuretic or pressor activities from uremic urine

It is our purpose to isolate and characterize the putative “Natriuretic Hormone”, ostensibly responsible for ECF homeostasis, as well as identify endonenous pressors and compounds that induce prolonged natriuresis; we report here our initial progress in this area. Large volumes of pooled urine collected from uremic patients were fractionated, and the resulting isolates were evaluated for in vivo natriuretic and pressor effects and Na +/K +-ATPase inhibitory activity in renal cells. The purification steps involved ultrafiltration to obtain materials of less than 3000 da, gel filtration, and sequential reversed-phase high performance liquid chromatography (HPLC). After each HPLC step, the fractions were evaluated for their ability to elicit significant natriuresis and/or influence mean arterial pressure in the normal conscious female rat. Each fraction was also assayed for its ability to inhibit Na +/K +-ATPase as determined by the inhibition of 86Rb + uptake into MDBK renal cells. While several of the fractions elicited profound natriuresis and/or pressor activity and other fractions inhibited Na +/K +-ATPase, there was no correlation among the activities in individual fractions. We have concluded that this plethora of bioactivities is responsible for much of the confusion and multiplicity of crude isolates claimed to be the putative hormone. Presently we are attempting to purify each of these activities to chemical homogeneity for structure determination.

Effect of hypothalamic lesions on interaction of centrally administered ANF and the circulating sodium-pump inhibitor.

We investigated the possible central interaction of atrial natriuretic factor (ANF) and an endogenous Na+, K(+)-ATPase (Na-pump) inhibitor in normal rats. Release of an endogenous Na-pump inhibitor associated with deoxycorticosterone acetate-salt hypertension may be regulated in the anteroventral third ventricle (AV3V) area of the CNS. We reported earlier that bolus injection of synthetic 26-amino acid ANF (Arg101-Tyr126, 6 micrograms/250 g rat) into the lateral brain ventricle (ICV) promotes the appearance in the plasma of a Na-pump inhibitor in rats. To determine whether the AV3V area of the brain is involved in the ICV effect of ANF, we introduced electrolytic lesions in this area. This treatment abolished the appearance of the Na-pump inhibitor after intraventricular injection of ANF. To further localize the area and the pathways involved in the interaction of ANF and the Na-pump inhibitor, we produced bilateral medial coronal knife cuts designed to transect the medially coursing pathway through the periventricular tissue of the AV3V region between the level of the medial preoptic area and the anterior hypothalamic nuclei. These knife cuts also abolished the appearance of the Na-pump inhibitor after ICV injection of ANF. Our data to date indicate that centrally administered ANF promotes the appearance of a Na-pump inhibitor in the plasma. A central site of interaction between ANF and the Na-pump inhibitor appears to be the AV3V area and a medial pathway coursing caudally from the AV3V region.

Properties of the purified hypothalamic pituitary NA/K-ATPase inhibitor.

Previously we described the isolation and final purification of an endogenous sodium-pump inhibitor from the CNS, mainly from bovine hypothalamus and pituitary. The purification protocol consisted of lipophilic chromatography, followed by lipid extraction, and semipreparative and analytical reverse-phase high-pressure liquid chromatography. The bioassays used were in vitro Na+/K(+)-ATPase inhibition, and 3H-ouabain displacement from its specific binding site in the enzyme structure, as well as inhibition of 86Rb uptake from human red blood cells. We have obtained, from both tissues, a low-molecular-weight, nonpeptidic, nonlipidic substance that elutes as a single peak highly pure according to criteria of coincidence of its spectra properties. When rechromatographed in two different chromatographic systems, the same homogeneous peak is obtained suggesting complete purity. This pure substance can be isolated from other bovine tissues as well as from human plasma and human placenta. It shows a very distinctive fluorescence spectrum and it behaves as a potent inhibitor of the Ca2+ pump of synaptosomal plasma membrane.

A Novel Endogenous Sodium-Pump Inhibitor in Pig Urine

To clarify the chemical nature and pathophysiological significance of an endogenous, specific Na-pump inhibitor, extracts of various tissues have been examined. The activities were extracted by an Amberlite XAD-2 adsorbent and fractionated by high-performance liquid chromatography (HPLC) on an ODS column. Among many tissues and fractions tested, the extract of pig urine has been found to contain a large amount of 86Rb uptake inhibitory activities. The strongest inhibitory activity for 86Rb influx into erythrocytes also exhibited a cross-reactivity with anti-ouabain antibodies. This activity has been purified to homogeneity using five steps of reverse-phase HPLC. Although most of the dose-response curves for this purified Na-pump inhibitor, designated as uroxin, in the various assay systems paralleled those of ouabain and the inhibitor purified from bovine adrenal glands (designated as adrexin C), the cross-reactivity curve with anti-ouabain antibodies did not. The retention time of uroxin on an ODS HPLC column was also different from that of digoxin, ouabain, or adrexin C. 1H nuclear magnetic resonance spectroscopic study suggests that uroxin is a novel Na-pump inhibitor that is structurally different from any of the known cardiotonic steroids or the substances previously reported to exhibit Na-pump inhibitory activity. Thus, uroxin may be a new type of endogenous regulator for the Na pump.

Physiological Significance of Na+/K+-ATPase Activity in the Central Nervous System and Endogenous Sodium-Pump Inhibitors in the Neural Regulation of Arterial Blood Pressure

In anesthetized Sprague-Dawley rats, cerebrolateral ventricular administration of potassium chloride solutions (KCl, 0.375-1.25 mumol, i.c.v.) produced concentration-dependent reductions in the arterial blood pressure and heart rate. These responses were significantly attenuated by prior i.c.v.-administration ouabain, a selective inhibitor of the Na+ pump, and by endothelin (ET-1), an endogenous peptide that is present in the CNS, suggesting that this peptide may participate in the neural regulation of arterial pressure via modulation of Na(+)-pump activity. Although both acute fluid volume expansion and/or osmotic stimulus have been shown to facilitate the release of the endogenous Na(+)-pump inhibitor(s) into the circulation, only volume expansion significantly attenuated the cardiovascular effects of i.c.v. potassium chloride. These observations collectively suggest that the Na+, K(+)-ATPase activity in CNS and Na(+)-pump inhibitors may play a significant role in the central regulation of arterial pressure under certain physiological conditions.

Cell volume changes upon sodium pump inhibition in Helix aspersa neurones.

1. Identified neurones of the suboesophageal ganglia of Helix aspersa were loaded with tetramethylammonium (TMA+). Experimentally induced changes in cell water volume and membrane potential were measured continuously by monitoring changes in intracellular [TMA+] using ion-sensitive double-barrelled microelectrodes. The technique allowed measurements of cell water volume changes of less than 5%. 2. Exposure to hyperosmotic (up to +24%) or hyposmotic (up to about -10%) solutions caused reversible decreases and increases in cell water volume respectively, which agreed with near-ideal osmometric behaviour. Upon exposure to hyposmotic solutions whose osmolality was decreased by 30-40%, the cell water volume increased to maximum values below those expected for ideal osmometric behaviour and exhibited partial regulatory volume decrease. 3. The sodium pump was inhibited in twenty identified neurones by sustained exposure to 1 mM ouabain. In every case ouabain caused cell membrane depolarization, as expected for inhibition of an electrogenic sodium pump. 4. Upon pump inhibition most cells (n = 14) shrank by up to 13% of their initial water volume. In five of these cells, shrinkage was preceded by one or more short-lived swelling phases. In two other neurones short-lived swelling was followed by cell volume recovery without appreciable shrinkage. In four out of the twenty cells, there were no measurable volume changes. 5. The lack of an initial swelling phase in the cells that shrank, as well as the absence of detectable volume changes in some of the neurones, was not due to loss of ion-selective electrode sensitivity since predictable changes in cell volume elicited by osmotic challenges were monitored in the same cells. 6. It is concluded that neurones can be endowed with ouabain-insensitive mechanisms of volume control, whose activation following Na+ pump inhibition prevents them from short-term swelling and lysis.

Inhibition of sodium pump by bepridil: An in vitro and microcalorimetric study

The effects of diltiazem, verapamil, bepridil, nicardipine and nifedipine were studied in vitro on Na +,K +-ATPase from dog kidney (EC 3.6.1.37). Except diltiazem, all the drugs tested showed an inhibitory effect on Na +,K +-ATPase activity in a dose-dependent manner. Among these drugs bepridil is far more effective than the others ( ic 50 ≈ 10 −4 M). Competition studies showed that bepridil acted in a non-competitive manner with the ATP-Mg 2+ complex and in a partially competitive manner with K +. Since ouabain acted similarly under the same experimental conditions, we tested the interaction of bepridil and ouabain on Na +, K +-ATPase. With low doses of ouabain, the enzyme inhibition corresponded to a potentiated synergy of the two drugs. We then studied the action of bepridil on the sodium pump activity of intact red blood cells by an ex vivo microcalorimetric technique. At 10 −5 M bepridil caused a significant decrease in sodium pump activity (33 ± 8%).